The guanidine (Gu) induced unfolding of intact alpha1-PI proteinase inhibitor (alpha 1-PI) shows an intermediate state at 1.5 M Gu. We initially observe several intermediates in 1.5 M Gu that slowly give rise to a final mixture containing monomeric and several polymeric intermediates. We have shown that this is an equilibrium mixture since its composition is the same whether obtained from native (O M Gu) or fully unfolded (greater than or equal to 5.5 M Gu) protein at constant final total protein concentration and since the reaction was demonstrated to be reversible. Furthermore, increase in total protein concentration results in increased levels of polymeric intermediates in the equilibrium mixture. Refolding from 1.5 M Gu after equilibration by dilution with buffer, by one-step dialysis, and by nine-step dialysis results in corresponding increasing levels of folded polymers, respectively. Starting with highly purified, intact alpha 1-PI, the form which shows inhibitory activity, we plan to characterize the intermediates in 1.5 M Gu in terms of their structure and the mechanism, kinetics, and thermodynamics of their formation. In addition, we plan to study the kinetics of refolding from 1.5 M Gu. Differential scanning calorimetry (DSC) studies of human albumin (HA) determined as a function of scan rate and protein concentration demonstrate that by extrapolating to zero scan rate (condition of true equilibrium), the denaturation temperature is independent of protein concentration. This result suggests that even though these thermally induced unfolding reactions are not repeatable (due to aggregation), they are microscopically reversible thereby justifying the application of reversible thermodynamics. Thus, the thermal stabilization of HA by the series of saturated, straight chain fatty acid anions (C 1 through C10) was analyzed in order to extract information concerning the affinity and stoichiometry of binding each of these ligands to both the folded and unfolded forms of HA. This study also reveals the existence of a class of sterically restricted binding sites accessible to only the shortest chain fatty acid anions (C1 through C3). HA consists of a single polypeptide chain but is comprised of three homologous domains. By thermally denaturing HA, we have identified two extreme forms of the protein by DSC. Under physiological conditions, the heat denaturation of albumin occurs in a highly cooperative manner, i.e. a single almost ideal two-state transition is observed, whereas at lower ionic strength (i.e. lower halide ion concentration), the denaturation becomes highly non-cooperative, i.e. two, well separated, major transitions occur. The binding of halide anion appears to promote the conversion of the protein from a form that unfolds non-cooperatively to one that denatures in a highly cooperative way. The relative efficiency of halide ions in promoting this conversion is I- > Br- > Cl-. To obtain additional information concerning the domains and interactions stabilizing the protein, we may look at urea induced denaturation at low ionic strength.